Classic cadherins, including epithelial (E)- and neural (N)-cadherins, are major cell-cell adhesion receptors involved in the development, maintenance and function of most tissues, including the nervous system, epithelia and endothelia. In addition, cadherins play important roles in cell signaling, proliferation and differentiation. In cadherin-based adherens junctions (CAJ), the extracellular domains of transmembrane cadherins promote cell-cell adhesion by engaging in Ca++-dependent homophilic interactions, while the cytoplasmic domains are linked to the actin cytoskeleton via α- and β-catenins. Post-translational regulation of cadherin adhesive activities, including proteolytic processing of cadherins and disassembly of CAJ, play crucial roles in rapid changes in cell adhesion, signaling and apoptosis, but the molecular mechanisms involved in cadherin processing and CAJ disassembly remain mostly unknown.
Presenilin-1 (PS1) is a polytopic transmembrane protein involved in most cases of early-onset familial Alzheimer's disease (FAD). Cellular PS1 is cleaved to yield an N-terminal (PS1/NTF) and a C-terminal (PS1/CTF) fragment. Following cleavage, the resultant PS1 fragments form a stable 1:1 heterodimer which binds to the cytoplasmic juxtamembrane region of E-cadherin (Baki et al., (2001) Proc. Natl. Acad. Sci. USA, 98:2381-2386). PS1 is found in the ER-Golgi system, but upon formation of cell-cell contacts PS1 concentrates at intercellular sites at the cell surface where it forms complexes with the CAJ. In addition to E-cadherin, PS1 forms complexes with N-cadherin and it has been localized at synaptic sites. Recently it was reported that PS1 regulates a γ-secretase cleavage of both APP and Notch receptor and stimulates Aβ-production (Herreman et al., (2000) Nat. Cell Biol. 2:461-2).
In the brain, PS1 forms complexes with N-cadherin (Georgakopoulos et al. (1999) Mol. Cell. 4: 893-902), a type I transmembrane protein and a member of the classic cadherin family of Ca++-dependent cell adhesion factors (Gumbiner (1996) Cell 84: 345-357). Both proteins are expressed in neurons and have been found at the synapse (Georgakopoulos et al. (1999); Uchida et al. (1996) J. Cell. Biol. 135: 767-779). N-cadherin homophilic interactions are thought to play an important role in holding together pre- and post-synaptic membranes (Fannon and Colman (1996) Neuron 17: 423-434) and N-cadherin has been shown to undergo molecular changes in response to synaptic activity (Tanaka et al. (2000) Neuron 25: 93-107). Furthermore, N-cadherin promotes axonal outgrowth and regulates synaptogenesis and long term potentiation (LTP) (Goda (2002) Neuron 35: 1-3).
PS1 is important for the γ-secretase cleavages of the amyloid precursor protein (APP), which result in the production of the Aβ peptide of Alzheimer's disease (AD) (De Strooper et al. (1998) Nature 391: 387-390). In addition to the classic γ-secretase cleavages of APP defined by the C-terminus of various Aβ species, the PS1/γ-secretase system promotes the γ-secretase-like, or ε-cleavage (Weidemann et al. (2002) Biochemistry 41: 2825-2835) of several type I transmembrane proteins, including APP, Notch1 receptor, E-cadherin and CD44. Although this cleavage is also sensitive to γ-secretase inhibitors, it takes place further downstream from the amyloidogenic γ-secretase cleavages at a site closer to the membrane/cytoplasm interface than the γ-cleavages (De Strooper et al. (1999) Nature 398: 518-522). It has been discovered in accordance with the present invention that in certain cases, like E-cadherin (see FIG. 1), the ε-cleavage is greatly stimulated by calcium imbalance or apoptosis. The ε-cleavage results in the release of soluble cytosolic peptides containing the intracellular domains (ICDs) of the cleaved substrate proteins. Some of these peptides have been shown to migrate to the nucleus where they may act as regulators of gene expression (for reviews see Ebinu and Yankner (2002) Neuron 34: 499-502; Fortini (2002) Nat Rev. Mol. Cell. Biol. 3: 673-684).
Transcriptional coactivator CBP (CREB binding protein) interacts with and regulates the activities of a multitude of signal-responsive transcription factors and may thus integrate converging gene-regulatory pathways (Goodman and Smolik (2000) Genes Dev. 14: 1553-1577). CBP acts as a scaffold that facilitates recruitment of additional transcriptional modulators on the basal transcriptional complex. In addition, CBP has an intrinsic histone acetyltransferase (HAT) activity that may be used to regulate transcription by acetylating chromatin (Bannister and Kouzarides (1996) Nature 384: 641-643). CBP regulates many physiological processes including cell growth, differentiation, and apoptosis. Changes in CBP activities are associated with a large number of developmental, neurodegenerative and mental retardation conditions, including the human Rubenstein-Taybi syndrome, and overexpression of Drosophila CPB can lead to neurodegeneration suggesting that cellular levels of CBP are tightly regulated. CBP is a coactivator of transcription factor CREB (cyclic AMP response element binding protein) that regulates the expression of a variety of genes that contain CREs (cyclic AMP response elements) in their promoters. CREB is implicated in a number of cellular processes and diseases (Mayr and Montminy (2001) Nat. Rev. Mol. Cell. Biol. 2: 599-609) and CREB-dependent gene expression is critical for the function and plasticity of the nervous system (Lonze and Ginty (2002) Neuron 35: 605-523) including long-term memory and learning in both vertebrates and invertebrates (Kandel (2001) Science 294: 1030-1038). Stimulation of CREB-mediated transcription requires phosphorylation at CREB-Ser133, an event that leads to the recruitment of CBP and stimulation of transcription (Chrivia et al. (1993) Nature 365: 855-859).
In accordance with the present invention it has been discovered that apoptosis or Ca++ influx stimulates a PS1/γ-secretase-like cleavage of E-cadherin. It has been further discovered that PS1 binding to E-cadherin is required for PS1/γ-secretase-like cleavage of E-cadherin. This cleavage results in the release of the cytoplasmic sequence of E-cadherin, β-catenin and α-catenin to the soluble cytosol, thus facilitating disassembly of cadherin-based adherens junctions. It has further been discovered that peptides based upon the PS1 binding site of cadherin inhibit γ-secretase activity, and are thus useful for inhibiting amyloid formation. In addition, it has been discovered that PS1 promotes a γ-secretase-like, or ε-cleavage of N-cadherin. This cleavage results in the production of a soluble intracellular domain (ICD) fragment termed N-Cad/CTF2. This peptide fragment binds CBP and sequesters it to the cytoplasm thus decreasing nuclear CBP and suppressing CREB-mediated transcription.